本文選題：腎細(xì)胞癌 + VHL　； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究目的:絕大多數(shù)腎臟惡性腫瘤的組織學(xué)類型是腎透明細(xì)胞癌。約75%-80%的腎透明細(xì)胞癌患者中存在染色體缺失、基因突變及啟動(dòng)子甲基化導(dǎo)致的VHL等位基因失活表達(dá)缺失。很多研究也發(fā)現(xiàn)了VHL基因缺失、表達(dá)失活在腎透明細(xì)胞癌的發(fā)生及進(jìn)展過程中發(fā)揮著非常重要的作用。氧存在時(shí)VHL蛋白(p VHL)呈遞缺氧誘導(dǎo)因子HIF-α并與之結(jié)合使其通過泛素連接酶-蛋白酶體途徑降解,使HIF-α分子在細(xì)胞中處于合成與降解的動(dòng)態(tài)平衡。腎細(xì)胞癌細(xì)胞系786-O細(xì)胞中VHL基因表達(dá)缺失,細(xì)胞內(nèi)HIF-2α不能被識(shí)別并降解呈現(xiàn)持續(xù)高表達(dá)的狀態(tài),進(jìn)一步誘導(dǎo)下游百余種靶基因(HRGs)的表達(dá)。課題組前期實(shí)驗(yàn)中通過裸鼠體內(nèi)成瘤的實(shí)驗(yàn)證實(shí)了HIF-2α對(duì)腎癌細(xì)胞系786-O成瘤的重要作用,但HIF下游靶基因?qū)δI癌細(xì)胞的生長則表現(xiàn)為促進(jìn)、抑制或沒影響等不同情況。通過基因序列表達(dá)譜分析發(fā)現(xiàn)乳腺癌缺失基因-1(DBC1)及斯鈣素蛋白-1(STC1)屬于被HIF-2α誘導(dǎo)表達(dá)升高的下游靶基因,而且檢索文獻(xiàn)發(fā)現(xiàn)DBC1、STC1在多種惡性腫瘤均發(fā)揮著重要的作用,但它們對(duì)腎細(xì)胞癌腫瘤形成的作用及機(jī)制并沒有系統(tǒng)深入的研究。本研究的目的在于驗(yàn)證DBC1、STC1會(huì)被HIF-2α誘導(dǎo)表達(dá),屬于HIF-2α下游基因,并探討它們對(duì)腎細(xì)胞癌腫瘤形成的作用及可能機(jī)制,旨在尋找潛在的腎細(xì)胞癌靶向治療的新靶點(diǎn)及診斷或預(yù)后新的生物學(xué)標(biāo)志物,優(yōu)化腎癌治療的個(gè)體化方案,改善晚期腎癌患者的預(yù)后。研究方法:1.培養(yǎng)本室構(gòu)建并保存的四種人類腎透明細(xì)胞癌細(xì)胞786-O(VHL-/-、VHL+/+、VHL-/-HIF-2αsh566、VHL+/+HIF-2α-d PA),使用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)DBC1、STC1在四種細(xì)胞系中m RNA水平的表達(dá),驗(yàn)證其是否屬于HIF-2α誘導(dǎo)表達(dá)升高的基因;2.通過使用慢病毒攜帶sh RNA DBC1、sh RNA STC1及SCR感染786-O(VHL-/-)腎癌細(xì)胞分別沉默DBC1、STC1的表達(dá)及對(duì)照組細(xì)胞,嘌呤霉素篩選得到穩(wěn)定轉(zhuǎn)染的細(xì)胞系,使用Western Blot檢測(cè)相應(yīng)細(xì)胞中DBC1、STC1的表達(dá)情況;3.MTT分別檢測(cè)陰性對(duì)照的786-O(VHL-/-)細(xì)胞和沉默DBC1表達(dá)及沉默STC1表達(dá)的786-O(VHL-/-)細(xì)胞的體外增殖情況;4.分別使用沉默DBC1表達(dá)及沉默STC1表達(dá)的786-O(VHL-/-)細(xì)胞注入裸鼠一側(cè)皮下作為實(shí)驗(yàn)組,對(duì)側(cè)皮下注入攜帶對(duì)應(yīng)陰性對(duì)照的786-O(VHL-/-)細(xì)胞作為對(duì)照組來進(jìn)行體內(nèi)成瘤實(shí)驗(yàn),8周后處死裸鼠取出皮下腫瘤組織,進(jìn)行稱重后統(tǒng)計(jì)并比較兩組實(shí)驗(yàn)組及對(duì)照組裸鼠移植瘤生長情況。結(jié)果:1.DBC1及STC1在786-O(VHL+/+、VHL-/-HIF-2αsh566)細(xì)胞的表達(dá)情況低于786-O(VHL-/-、VHL+/+HIF-2α-d PA)細(xì)胞中的表達(dá)情況,表明DBC1和STC1屬于HIF-2α的下游靶基因,能夠被HIF-2α誘導(dǎo)而表達(dá)升高。2.MTT實(shí)驗(yàn)結(jié)果表明沉默DBC1在786-O(VHL-/-)細(xì)胞中的表達(dá)沒有對(duì)腎癌細(xì)胞的體外增殖產(chǎn)生影響,沉默STC1在786-O(VHL-/-)細(xì)胞中的表達(dá)則抑制腎癌細(xì)胞的體外增殖。3.沉默DBC1和STC1在786-O VHL-/-細(xì)胞中的表達(dá)后裸鼠移植瘤成瘤能力明顯小于對(duì)照,DBC1組和STC1組中實(shí)驗(yàn)組與對(duì)照組的差異均具有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論:1.DBC1、STC1在人類腎透明細(xì)胞癌細(xì)胞786-O中被HIF-2α誘導(dǎo)表達(dá)升高,是HIF-2α的下游靶基因。2.DBC1的表達(dá)對(duì)腎癌細(xì)胞體外增殖沒有影響,STC1的表達(dá)促進(jìn)腎癌細(xì)胞的體外增殖;DBC1和STC1都明顯促進(jìn)裸鼠移植瘤成瘤,提示它們?cè)谀I癌中是促癌因素。沉默DBC1及STC1表達(dá)抑制了786-O(VHL-/-)腎癌細(xì)胞系裸鼠移植瘤的形成,表明DBC1、STC1是可能的腎癌分子靶向治療的新靶點(diǎn)。3.VHL-HIF-HRGs調(diào)節(jié)通路是促進(jìn)腎細(xì)胞癌發(fā)生發(fā)展的關(guān)鍵,但仍有一大批被HIF-2α誘導(dǎo)表達(dá)升高的靶基因在腎細(xì)胞癌腫瘤形成的過程中發(fā)揮的作用還沒有被系統(tǒng)得證實(shí),它們可能通過參與不同的信號(hào)通路,發(fā)揮著對(duì)腎細(xì)胞癌發(fā)生發(fā)展相關(guān)的作用。對(duì)這些基因進(jìn)行進(jìn)一步的篩選和驗(yàn)證有助于深入全面地了解腎細(xì)胞癌腫瘤發(fā)生發(fā)展的機(jī)制,并可以探索更多腎癌分子靶向治療的新靶點(diǎn)。
[Abstract]:Objective: the histologic type of the vast majority of renal malignant tumors is clear cell carcinoma of the kidney. There are chromosomal deletion, gene mutation and promoter methylation of VHL allele inactivation in about 75%-80% of renal clear cell carcinoma. Many studies have also found VHL gene deletion and inactivation in renal clear cell carcinoma. When oxygen exists, the VHL protein (P VHL) presents hypoxia inducible factor HIF- alpha and combines with the ubiquitin ligase proteasome pathway to make the HIF- alpha molecule in the cell in the dynamic equilibrium of synthesis and degradation. The expression of VHL gene in the cell line of renal cell carcinoma cell line 786-O is deficient. The HIF-2 alpha in the cell can not be identified and degraded to present a continuous high expression state, which further induces the expression of more than 100 target genes (HRGs) in the lower reaches of the group. In the early experiments of the group, the tumor formation in nude mice confirmed the important role of HIF-2 alpha on the tumor cell line 786-O in the renal cell line, but the target gene of the downstream HIF on the growth of renal cell carcinoma cells -1 (DBC1) and -1 (STC1) are the downstream target genes which are elevated by HIF-2 alpha induced expression by gene sequence expression spectrum analysis, and the retrieval literature found that DBC1, STC1 plays an important role in various malignant tumors, but they are to renal cells. The role and mechanism of cancer formation have not been systematically studied. The purpose of this study is to verify that DBC1, STC1 can be induced by HIF-2 alpha, belongs to the HIF-2 alpha downstream gene, and to explore the role and possible mechanism of them on the formation of renal cell carcinoma, aiming to find new targets and diagnosis or prognosis of potential treatment of renal cell carcinoma. A new biological marker to optimize the individualized regimen for the treatment of renal carcinoma to improve the prognosis of patients with advanced renal cancer. 1. to cultivate four kinds of human renal cell carcinoma cell 786-O (VHL-/-, VHL+/+, VHL-/-HIF-2 a sh566, VHL+/+HIF-2 alpha -d PA) constructed and preserved in this room, and detect DBC1 with real-time fluorescent quantitative PCR technique, STC1 in four kinds of cells The expression of M RNA level in the system is to verify whether it belongs to the gene of elevated HIF-2 alpha induced expression; 2. by using lentivirus to carry sh RNA DBC1, sh RNA STC1 and SCR infected 786-O (VHL-/-) kidney cancer cells are silent, and the cells of the control group and purinamycin are screened for the stable transfected cell lines. The expression of DBC1 and STC1 in the cells; 3.MTT detection of negative control 786-O (VHL-/-) cells and silent DBC1 expression and the proliferation of 786-O (VHL-/-) cells expressing STC1 expression, respectively. 4. cells were injected subcutaneously into the nude mice by silencing DBC1 expression and silent STC1 expression as experimental group, subcutaneous injection of the contralateral side 786-O (VHL-/-) cells with negative control were used as the control group to carry out the tumor formation experiment in the body. After 8 weeks, the nude mice were killed and the subcutaneous tumor tissues were taken out. After weighing, the growth of the transplanted tumor in the two groups and the control group was compared. Results: the expression of 1.DBC1 and STC1 in 786-O (VHL+/+, VHL-/-HIF-2 alpha sh566) cells was low. The expression in 786-O (VHL-/-, VHL+/+HIF-2 alpha -d PA) cells indicates that DBC1 and STC1 belong to the downstream target gene of HIF-2 a, which can be induced by HIF-2 alpha and expressed in.2.MTT. The expression of.2.MTT in.2.MTT shows that the expression of silent DBC1 in 786-O cells does not affect the proliferation of renal cell carcinoma cells. The expression of.3. inhibited the proliferation of renal cell carcinoma cells in vitro and the expression of DBC1 and STC1 in 786-O VHL-/- cells was significantly less than that of the control. The difference between the experimental group and the control group in the DBC1 and STC1 groups was statistically significant (P0.01). Conclusion: 1.DBC1, STC1 is HIF-2 alpha in the 786-O of human renal cell carcinoma cells. The expression of.2.DBC1, the downstream target gene of HIF-2 alpha, has no effect on the proliferation of renal cancer cells in vitro. The expression of STC1 promotes the proliferation of renal cancer cells in vitro, and both DBC1 and STC1 obviously promote the tumor formation in nude mice, suggesting that they are cancer promoting factors in renal carcinoma. The silence of DBC1 and STC1 inhibits the 786-O (VHL-/-) renal cell line. The formation of xenografts in nude mice shows that DBC1, STC1 is a possible new target of molecular targeting therapy for renal cancer,.3.VHL-HIF-HRGs regulation pathway is the key to promote the development of renal cell carcinoma, but a large number of target genes which are induced by HIF-2 alpha induced expression have not been systematically confirmed by the role of the target gene in the process of renal cell carcinoma. They may play a role in the development of renal cell carcinoma by participating in different signaling pathways. Further screening and verification of these genes will help to understand the mechanism of the development of renal cell carcinoma and explore more new targets for molecular targeting therapy of renal cell carcinoma.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.11

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本文編號(hào)：1959247